State Key Laboratory for Animal Disease Control and Prevention, South China Agricultural University, Guangzhou, China.
School of Biology and Agriculture, Shaoguan University, Shaoguan, China.
PLoS Negl Trop Dis. 2024 Oct 3;18(10):e0012569. doi: 10.1371/journal.pntd.0012569. eCollection 2024 Oct.
Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. The C. parvum genome contains 22 genes encoding insulinase-like M16 proteases (INS) with diverse structures and sequences, suggesting that members of the protein family may have distinct biological functions in the life cycle of parasites. Here, we investigated the role of INS15 and INS16, two proteases encoded by neighboring genes with high sequence identity, in the growth and development of C. parvum in vivo and in vitro.
METHODOLOGY/PRINCIPAL FINDINGS: INS15 and INS16 genes were tagged and knocked out using CRISPR/Cas9 technology in C. parvum IIdA20G1-HLJ isolate. The expression of INS15 and INS16 was determined by immunofluorescence analysis and immunoelectron microscopy. The effect of depletion of INS15 and INS16 on parasite growth and pathogenicity were assessed on HCT-8 cells and in interferon-γ knockout mice. Endogenous tagging showed that INS15 and INS16 expressed in the oocyst, trophozoite, meront and female gametes. INS15 also expressed in male gamonts, while INS16 was not detected in the male gamonts. Although depletion of the INS15 or INS16 gene affected late development of C. parvum in vitro, only depletion of INS15 significantly reduced parasite burden in infected mice. Mice infected with the INS15-depleted strain had reduced clinical signs, body weight, intestinal villus length to crypt height ratio, and survival time compared to infected with the tagging mutant.
CONCLUSIONS/SIGNIFICANCE: The results of this study indicate that INS15 is mainly involved in the late development of C. parvum. Depletion of this gene attenuates the pathogenicity of this important zoonotic parasite.
微小隐孢子虫是一种常见的原生动物病原体，可导致人类和动物出现中度至重度腹泻。微小隐孢子虫基因组包含 22 个基因，这些基因编码结构和序列多样的胰岛素样 M16 蛋白酶（INS），表明该蛋白家族的成员在寄生虫的生命周期中可能具有不同的生物学功能。在这里，我们研究了由高度同源的邻近基因编码的蛋白酶 INS15 和 INS16 在体内和体外微小隐孢子虫生长和发育中的作用。
方法/主要发现：使用 CRISPR/Cas9 技术在微小隐孢子虫 IIdA20G1-HLJ 分离株中对 INS15 和 INS16 基因进行标记和敲除。通过免疫荧光分析和免疫电子显微镜确定 INS15 和 INS16 的表达。在 HCT-8 细胞和干扰素-γ基因敲除小鼠中评估 INS15 和 INS16 耗竭对寄生虫生长和致病性的影响。内源性标记表明 INS15 和 INS16 在卵囊、滋养体、裂殖体和雌性配子体中表达。INS15 也在雄性配子体中表达，而 INS16 未在雄性配子体中检测到。尽管 INS15 或 INS16 基因的缺失会影响微小隐孢子虫在体外的晚期发育，但只有 INS15 的缺失会显著降低感染小鼠中的寄生虫负担。与感染标记突变体相比，感染 INS15 缺失株的小鼠临床症状、体重、肠绒毛长度与隐窝高度比和存活时间减少。
结论/意义：本研究结果表明，INS15 主要参与微小隐孢子虫的晚期发育。该基因的缺失可减弱这种重要的人畜共患病寄生虫的致病性。
